Phosphate analogues are compounds chemically related to orthophosphate, but having chemical substitutions for one or two oxygen atoms. These substitutions result in unique changes in atomic structure, the number and acidity of titratible protons, and in the nuclear magnetic resonance (NMR) spectrum of each compound. These unique characteristics will be exploited to study by means of NMR spectroscopy two areas of human erythrocyte physiology: membrane anion transport and energy metabolism. Anion transport will be studied by simultaneous observation of intracellular and extracellular resonances, either naturally occurring as a result of pH sensitive chemical shifts, or induced by means of addition of an extracellular paramagnetic shift reagent. Membrane transport of rapidly exchanging compounds will be studied by saturation-transfer methods. Concentration and pH dependence of the transport mechanism will be studied, and net anion conductance will be measured using ionophores and inhibitors of the anion transport protein. These transport characteristics will be compared to the titratible charge model for anion transport. Since these compounds bear chemical similarities to orthophosphate, their effects on red cell energy metabolism will be studied, by monitoring glucose utilization, lactate production, intracellular pH, and synthesis of organic phosphates (2,3-DPG and ATP). Phosphate analogue effects on nucleoside (inosine) energy metabolism will also be elucidated. In addition, the influence of analogues on the activity of phosphofructokinase (a major glycolysis control enzyme stimulated by orthophosphate) and glyceraldehyde phosphate dehydrogenase (the only glycolytic enzyme incorporating phosphate de novo into metabolic intermediates) will be studied. During metabolic studies evidence wil be sought for enzymatic hydrolysis of phosphate analogues, intracellular synthesis of fluorophosphate from F- and orthophosphate, and for enzymatic incorporation of phosphate analogues into glycolytic metabolites.